Feng Y, Roder H, Englander S W
Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia 19104-6059.
Biochemistry. 1990 Apr 10;29(14):3494-504. doi: 10.1021/bi00466a011.
Proton nuclear magnetic resonance assignments for reduced and oxidized equine cytochrome c show that many individual protons exhibit different chemical shifts in the two protein forms, reflecting diamagnetic shift effects due to structure change, and in addition contact and pseudocontact shifts that occur only in the paramagnetic oxidized form. To evaluate the chemical shift differences (delta delta) for structure change, we removed the pseudocontact shift contribution by a calculation based on knowledge of the electron spin g tensor. The g-tensor parameters were determined from the delta delta values of a large set (64) of C alpha H protons at well-defined spatial positions in the oxidized horse protein. The g-tensor calculation, when repeated using only 12 available C alpha H proton resonances for cytochrome c from tuna, proved to be remarkably stable. The largest principal value of the g tensor (gz) falls precisely along the ligand bond between the heme iron and methionine-80 sulfur, while gx and gy closely match the natural heme axes defined by the pyrrole nitrogens. The derived g tensor was then used together with spatial coordinates for the oxidized form to calculate the pseudocontact shift contribution (delta pc) to proton resonances at 400 identifiable sites throughout the protein, so that the redox-dependent chemical shift discrepancy, delta delta-delta pc, could be evaluated. Large residual changes in chemical shift define the Fermi contact shifts, which are found as expected to be limited to the immediate covalent structure of the heme and its ligands and to be asymmetrically distributed over the heme. Smaller chemical shift discrepancies point to a concerted change, involving residues 39-43 and 50-60 (bottom of the protein), and to other changes in the immediate vicinity of the heme ligands. Also, the three internal water molecules are implicated in redox sensitivity. The residues found to change are in good but not perfect agreement with prior X-ray diffraction observations of subangstrom redox-related displacements in the tuna protein. The chemical shift discrepancies observed appear in the main to reflect structure-dependent diamagnetic shifts rather than hyperfine effects due to displacements in the pseudocontact shift field. Although 51 protons in 29 different residues exhibit significant chemical shift changes, the general impression is one of small structural adjustments to redox-dependent strain rather than sizeable structural displacements or rearrangements.
还原型和氧化型马细胞色素c的质子核磁共振归属表明，许多单个质子在两种蛋白质形式中表现出不同的化学位移，这反映了由于结构变化引起的抗磁位移效应，此外还有仅在顺磁性氧化形式中出现的接触位移和赝接触位移。为了评估结构变化引起的化学位移差异（δδ），我们基于电子自旋g张量的知识通过计算去除了赝接触位移贡献。g张量参数由氧化马蛋白中定义明确的空间位置处的一大组（64个）CαH质子的δδ值确定。当仅使用金枪鱼细胞色素c的12个可用CαH质子共振重复进行g张量计算时，结果证明非常稳定。g张量的最大主值（gz）恰好沿着血红素铁与甲硫氨酸-80硫之间的配体键，而gx和gy与由吡咯氮定义的天然血红素轴紧密匹配。然后将推导得到的g张量与氧化形式的空间坐标一起用于计算蛋白质中400个可识别位点处质子共振的赝接触位移贡献（δpc），从而可以评估氧化还原依赖性化学位移差异δδ - δpc。化学位移的大的残留变化定义了费米接触位移，正如预期的那样，发现其仅限于血红素及其配体的直接共价结构，并在血红素上不对称分布。较小的化学位移差异表明存在协同变化，涉及残基39 - 43和50 - 60（蛋白质底部）以及血红素配体紧邻区域的其他变化。此外，三个内部水分子与氧化还原敏感性有关。发现发生变化的残基与之前对金枪鱼蛋白中亚埃级氧化还原相关位移的X射线衍射观察结果有较好但不完全一致的情况。观察到的化学位移差异主要似乎反映了结构依赖性抗磁位移，而不是赝接触位移场中位移引起的超精细效应。尽管29个不同残基中的51个质子表现出显著的化学位移变化，但总体印象是对氧化还原依赖性应变的小结构调整，而不是相当大的结构位移或重排。
